Humans and other warm-blooded animals can be afflicted by a number of bone-related disorders. Such disorders range from bone fractures, to debilitating diseases such as osteoporosis. While in healthy individuals bone growth generally proceeds normally and fractures heal without the need for pharmacological intervention, in certain instances bones may become weakened or may fail to heal properly. For example, healing may proceed slowly in the elderly and in patients undergoing treatment with corticosteroids (e.g., transplant patients). Osteoporosis is a condition in which bone hard tissue is lost disproportionately to the development of new hard tissue. Osteoporosis can generally be defined as the reduction in the quantity of bone, or the atrophy of skeletal tissue; marrow and bone spaces become larger, fibrous binding decreases, and compact bone becomes fragile. Another bone related disorder is osteoarthritis, which is a disorder of the movable joints characterized by deterioration and abrasion of articular cartilage, as well as by formation of new bone at the joint surface.
While a variety of treatments are available for such bone-related disorders, none of the treatments provide optimum results. One of the difficulties facing individuals who treat bone-related disorders is a lack of complete understanding of bone metabolism and of the bone-related disorders. A key to such understanding is identifying and characterizing each of the components involved in bone growth. Bone morphogenetic proteins (BMPs) have been demonstrated to play a role in bone formation and development (J. M. Wozney, Molec. Reproduct. and Develop., 32: 160-167 (1992)).
Furthermore, the role of BMPs may not be limited to their role in bone. The finding that the BMPs are found at significant concentrations in other tissues such as brain, kidney, stratified squamous epithelia, and hair follicle (N. A. Wall, M. Blessing, C. V. E. Wright, and B. L. M. Hogan, J. Cell Biol., 120: 493-502 (1993); E. Özkaynak, P. N. J. Schnegelsberg, D. F. Jin, G. M. Clifford, F. D. Warren, E. A. Drier, and H. Oppermann, J. Biol. Chem., 267: 25220-25227 (1992); K. M. Lyons, C. M. Jones, and B. L. M. Hogan, Trends in Genetics, 7: 408-412 (1991); V. Drozdoff, N. A. Wall, and W. J. Pledger, Proceedings of the National. Academy of Sciences, U.S.A., 91: 5528-5532 (1994)) suggests that they may play additional roles in development and differentiation. In support of this, BMPs have recently been found to promote nerve cell differentiation and to affect hair follicle formation (K. Basler, T. Edlund, T. M. Jessell, and T. Yamada, Cell, 73: 687-702 (1993); V. M. Paralkar, B. S. Weeks, Y. M. Yu, H. K. Kleinman, and A. H. Reddi, J. Cell Biol., 119: 1721-1728 (1992); M. Blessing, L. B. Nanney, L. E. King, C. M. Jones, and B. L. Hogan, Genes Dev., 7: 204-215 (1993)).
A BMP initiates its biological effect on cells by binding to a specific BMP receptor expressed on the plasma membrane of a BMP-responsive cell. A receptor is a protein, usually spanning the cell membrane, which binds to a ligand from outside the cell, and as a result of that binding sends a signal to the inside of the cell which alters cellular function. In this case, the ligand is the protein BMP, and the signal induces the cellular differentiation.
Because of the ability of a BMP receptor to specifically bind BMPs, purified BMP receptor compositions are useful in diagnostic assays for BMPs, as well as in raising antibodies to the BMP receptor for use in diagnosis and therapy. In addition, purified BMP receptor compositions may be used directly in therapy to bind or scavenge BMPs, thereby providing a means for regulating the activities of BMPs in bone and other tissues. In order to study the structural and biological characteristics of BMP receptors and the role played by BMPs in the responses of various cell populations to BMPs during tissue growth/formation stimulation, or to use a BMP receptor effectively in therapy, diagnosis, or assay, purified compositions of BMP receptor are needed. Such compositions, however, are obtainable in practical yields only by cloning and expressing genes encoding the receptors using recombinant DNA technology. Efforts to purify BMP receptors for use in biochemical analysis or to clone and express mammalian genes encoding BMP receptors have been impeded by lack of a suitable source of receptor protein or mRNA. Prior to the present invention, few cell lines were known to express high levels of high affinity BMP receptors which precluded purification of the receptor for protein sequencing or construction of genetic libraries for direct expression cloning. Availability of the BMP receptor sequence will make it possible to generate cell lines with high levels of recombinant BMP receptor for biochemical analysis and use in screening experiments.
The BMPs are members of the TGF-β superfamily. Other members of the TGF-β superfamily include TGF-β, activins, inhibins, Müllerian Inhibiting Substance, and the Growth and Differentiation Factors (GDFs). As expected, the receptors for various members of the TGF-β superfamily share similar structural features. Receptors of the TGF-β ligand superfamily are typically classified into one of two sub-groups, designated as type I and type II. The type I and type II receptors are classified as such based on amino acid sequence characteristics. Both the type I and type II receptors possess a relatively small extracellular ligand binding domain, a transmembrane region, and an intracellular protein kinase domain that is predicted to have serine/threonine kinase activity (Lin and Moustakas, Cellular and Molecular Biology, 40: 337-349 (1994); L. S. Mathews, Endocrine Reviews, 15: 310-325 (1994); L. Attisano, J. L. Wrana, F. López-Casillas, and J. Massagué, Biochimica et Biophysica Acta, 1222: 71-80 (1994)).
The type I receptors cloned to date belong to a distinct family whose kinase domains are highly related and share >85% sequence similarity (B. B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). The intracellular juxtamembrane region of the type I receptors is characterized by an SGSGSG motif 35-40 amino acids from the transmembrane region, and the carboxy terminus of these receptors is extremely short (B. B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, J. L. Wrana, F. López-Casillas, and J. Massagué, Biochimica et Biophysica Acta, 1222: 71-80 (1994)). The extracellular domain of the type I receptors contains a characteristic cluster of cysteine residues, termed the “cysteine box”, located within 25-30 amino acids of the transmembrane region, and another cluster of cysteine residues, termed the “upstream cysteine box”, located after the putative signal sequence (B. B. Koenig, et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, et al., Biochimica et Biophysica Acta, 1222: 71-80 (1994)).
In contrast to the type I receptors, the kinase domains of the type II receptors are only distantly related to one another. The SGSGSG motif found in type I receptors is not found in type II receptors. Also, the “upstream cysteine box” of type I receptors is not present in type II receptors. Furthermore, while all of the activin type II receptors contain a proline-rich sequence motif in the intracellular juxtamembrane region, there is no characteristic sequence motif that is common to all type II receptors (L. S. Mathews, Endocrine Reviews, 15: 310-325 (1994)). The length of the carboxy terminus of the type II receptors is considerably variable, with the longest known carboxy terminus being found in the BMP type II receptor, DAF-4 (M. Estevez, L. Attisano, J. L. Wrana, P. S. Albert, J. Massagué, and D. L. Riddle, Nature, 365: 644-49 (1993)), that was cloned from the nematode C. elegans. The extracellular domain of the type II receptors contains a single cysteine box located near the transmembrane region. Aside from the presence of the cysteine box, there is little sequence similarity amongst the extracellular domains of the type II receptors for TGF-β, activin, and BMPs.
Signaling by members of the TGF-β ligand superfamily requires the presence of both type I and type II receptors on the surface of the same cell (L. S. Mathews, Endocrine Reviews, 15: 310-325 (1994); L. Attisano, J. L. Wrana, F. López-Casillas, and J. Massagué, Biochimica et Biophysica Acta, 1222: 71-80 (1994)). The BMPs are members of the TGF-β ligand superfamily; given the high degree of structural similarity among these family members, it is expected that their receptors will be structurally and functionally related to the TGF-β and activin receptors. It is anticipated that, like the TGF-β and activin receptor systems (J. Massagué, L. Attisano, and J. L. Wrana, Trends in Cell Biology, 4: 172-178 (1994)), both a BMP type I receptor and a BMP type II receptor will be needed in order to transduce a BMP signal within a cell or tissue. Hence, there is a need for a mammalian type II BMP receptor kinase protein in addition to the type I receptors that have already been cloned.
Three distinct mammalian type I receptors have been reported for the BMPs: Bone Morphogenetic Protein Receptor Kinase-1 (herein referred to as “BRK-1”) (see U.S. Ser. No. 08/158,735, filed Nov. 24, 1993 by J. S. Cook, et al.; and B. B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)), ALK-2, and ALK-6. BRK-1 is the mouse homologue of ALK-3, which has also been demonstrated to bind BMP-4, as does ALK-6; ALK-2 binds BMP-7 (see P. ten Dijke, H. Yamashita, T. K. Sampath, A. H. Reddi, M. Estevez, D. L. Riddle, H. Ichijo, C. -H. Heldin, and K. Miyazono, J. Biological Chemistry, 269: 16985-16988 (1994)). It is also postulated that ALK-6 is the mouse homologue of the chicken receptor Bone Morphogenetic Protein Receptor Kinase-2 (herein referred to as “BRK-2”) (also referred to as RPK-1) (S. Sumitomo, T. Saito, and T. Nohno, DNA Sequence, 3: 297-302 (1993)). The rat homologue of BRK-1 has also been cloned, as BMPR-Ia (K. Takeda, S. Oida, H. Ichijo, T. Iimura, Y. Marnoka, T. Amagasa and S. Sasaki, Biochemical and Biophysical Research Communications, 204: 203-209 (1994)).
In co-pending application U.S. Ser. No. 08/334,179, filed Nov. 4, 1994 by Rosenbaum and Nohno, a novel mammalian BMP type II receptor (referred to as “BRK-3”) is described and claimed. Prior to the cloning of the BRK-3 receptor, the only type II receptor for BMP-2 and BMP-4, named DAF-4, was cloned from the nematode C. elegans (M. Estevez, L. Attisano, J. L. Wrana, P. S. Albert, J. Massagué, and D. L. Riddle, Nature, 365: 644-9 (1993)). Because of the large evolutionary distance between the nematode and mammals, it has not been possible to use the DAF-4 cDNA as a probe with which to clone the mammalian DAF-4 homologue. This implies that the DNA sequence of the mammalian type II receptor for BMPs is substantially divergent from that of DAF-4, and it was therefore necessary to clone a mammalian type II receptor for the BMPs.
The BMP receptor kinase protein BRK-3 of the co-pending application provides a mammalian type II receptor which enables the formation of a complex with a BMP type I receptor. This complex, which is described in detail below, is capable of binding BMPs with high affinity, and is therefore useful for identifying compounds having BMP receptor affinity. The complex of the present invention will also enable the formation of a high affinity complex that is competent for signaling a response to BMPs in concert with the mammalian type I receptor(s) for BMPs. The mammalian BMP receptor complex is therefore more relevant for the identification of novel compounds which interact with the BMP receptor, and which will be useful as therapeutic agents in humans and other mammals, than is a receptor complex that is composed of the nematode type II receptor and the mammalian type I receptor.